Validation of microbiological methods for use in the food industry
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Validation of Microbiological Methods for Use in the Food Industry. Brazilian Association for Food Protection 6 th International Symposium Sao Paulo, Brazil June 15 th , 2007. Introduction. Hundreds of new methods developed each year Pathogenic organisms Non-Pathogenic organisms

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Validation of Microbiological Methods for Use in the Food Industry

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Validation of Microbiological Methods for Use in the Food Industry

Brazilian Association for Food Protection

6th International Symposium

Sao Paulo, Brazil

June 15th, 2007


Introduction

  • Hundreds of new methods developed each year

    • Pathogenic organisms

    • Non-Pathogenic organisms

    • Detection

    • Identification

  • How do you know if you need a new method?

  • How do you decide if it is the right method for your purpose?


Introduction

  • Goal of methods evaluation is to find an innovative technology that will allow for quick and efficient detection and/or quanitation of pathogens and spoilage organisms


Performance Criteria

  • The Three S’s

    • Sensitivity

      • What is the sensitivity of current method

      • What degree of sensitivity is needed

    • Specificity

      • What is the false positive rate

      • What is the false negative rate

    • Speed

      • What is speed of current method (samples processed/day)

      • How quickly are results needed


Performance Criteria

  • Costs

    • What is cost of current method

    • What is cost of instrumentation

    • What is cost of disposables/reagents

    • What is the cost per test

  • Reagents

    • Prep time

    • Stability

    • Availability

    • Consistency (Quality Control)


Performance Criteria

  • Versatility

    • Product only

      • Variety of food matrixes

    • Environmental samples only

    • Pathogens only

    • Microorganisms only

      • Bacteria and/or Fungi

  • Acceptability of method by scientific community and/or Regulators

    • AOAC, AOAC-RI, USDA-FSIS, FDA, AFNOR


Performance Criteria

  • Vendor company reputation

    • First product on market

  • Training

    • Vendor provided training on site

    • How much, how long

  • Technical Service

    • Speed of service

    • Availability of service (24-7)

    • Service contract required


Technical Evaluation

  • Objective

  • Justification (benefit of method to company)

  • Acceptance Criteria

  • Material and Methods

    • Test Media/Conditions

  • Microorganisms

    • Genus, species, source

    • Inoculum preparation

  • Inoculation Procedure

  • Statistical Analysis

  • Results

  • Next Steps


Case Study #1 Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation


Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation

  • Objective: Determine validity of a 2 day yeast and mold method using DRB agar incubated at 30C or 35C

  • Justification: Reduced product holding time, resulting in significant cost savings to the plant

  • Acceptance Criteria: Recovery efficiencies must be equivalent to the current 5 day PDA method


Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation

  • Microorganisms:

    • Mold Cultures

      • A.niger, Penicillium spp., and Paecilomyces spp.

    • Yeast Cultures

      • Z.ballii, S.cerevisiae, and a plant isolate

  • Inoculum Preparation:

    • Organisms were harvested from aPDA plates by washing with sterile water

    • 1ml from each individual mold or yeast suspension was added to 20 mls DI water

      • Molds serially diluted

      • Yeast adjusted to a spec reading of 1.00, then serially diluted


Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation

  • Material and Methods:

    • product was inoculated with 100 cfu/g of target organisms

    • 0.1ml of inoculated product surface plated onto each media (aPDA, DRBA)

    • aPDA incubated at 25C

      • Counted at 3 and 5 days

    • DRBA incubated at 30C and 35C

      • Counted at 2, 3, 4, and 5 days


Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation

  • Statistical Analysis:An analysis of variance (AOV) was done to test if the total counts for DRB at 2 and 5 days was significantly different from aPDA at 5 days


Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation

  • Results:

    • DRB at 2 days-30C was statistically equivalent to aPDA at 5 days for mold recovery

      • Molds were pale in color; Penicillium spp. was white on DRB (green on aPDA). The other 2 test molds were pale yellow

    • Yeast counts on DRB at 30C were significantly lower than counts on aPDA at 2 and 5 days

    • Mold and Yeast counts were significantly lower on DRB at 35C vs. aPDA


Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation

  • Conclusion:

    • Due to overall decreased recovery of yeast and mold, and the mold visual observations; the Dichloran-Rose Bengal Agar Yeast and Mold recovery medium is not recommended.


Case Study #2 Rapid Check SalmonellaTest Kit Evaluation


Rapid Check SalmonellaTest Kit Evaluation

  • Objective: Determine validity of the Strategic Diagnostics Inc. Rapid Check antibody lateral flow method for the detection of Salmonella in comparison to the BAX PCR test method

  • Justification: Reduce testing cost, false positives rate and technician time


Rapid Check SalmonellaTest Kit Evaluation

  • Acceptance Criteria:

    • Speed; shorter time to results vs. PCR?

    • Sensitivity; greater or equivalent to PCR?

    • Specificity; greater or equivalent to PCR

  • Cost

    • Less than or equal to BAX PCR system

    • Cost per test

  • Versatility; food products only, environmental samples only, or both?


Rapid Check SalmonellaTest Kit Evaluation

  • Organisms and Inoculum Preparation:

    • A cocktail of 5 Salmonella spp.

    • A cocktail of 7 non-Salmonella spp.

      • E.coli (2), Citrobacter, Bacillus, Klebsiella, Enterobacter (2)

    • Individual cultures grown overnight in BHI at 35C

    • Salmonella strains pooled, diluted to 100cfu/ml

    • Non-Salmonella strains pooled, diluted to 1,000 cfu/ml


Rapid Check SalmonellaTest Kit Evaluation

  • Methods:

    • Inoculation of samples

      • With Salmonella

      • With non-Salmonella strains

      • With both

    • Pre-enrichment of samples

      • Traditional medium; Lactose for 24 hours

      • SDI medium for 5 hours

    • Secondary enrichment

      • Tetrathionate for 24 hours


Rapid Check SalmonellaTest Kit Evaluation

  • Methods (cont):

    • BAX PCR analysis

      • 3 hour re-growth

      • Cell lysis

      • 4-8 hour PCR cycle

    • SDI lateral flow assay

      • Load 150ul onto SDI cartridge

      • Develop for 10 minutes


Rapid Check SalmonellaTest Kit Evaluation

  • Results:

    • Sensitivity

      • Results were more consistent with SDI when recovering at the threshold level (1000 cfu/ml in the TT broth)

      • Equivalent results with both methods above the threshold level

      • SDI 5 hour pre-incubation media did not consistently support growth above the threshold level (acceptance criteria)

    • Specificity

      • No cross reactivity with non-Salmonella organisms with either method


Rapid Check SalmonellaTest Kit Evaluation

  • Results: Speed


Rapid Check SalmonellaTest Kit Evaluation

  • Conclusions:

    • SDI shown to be as sensitive as BAX-PCR

      • 5 hour medium not recommended

    • No cross reactivity observed with SDI

    • SDI gave results sooner than PCR

    • PCR has more steps, more prone to technician error

    • Some degree of subjectivity with SDI

    • SDI easier to use; 1 step inoculation of 1 single cartridge


Rapid Check SalmonellaTest Kit Evaluation

  • Conclusions:

    • SDI can be successfully used for food and environmental samples

    • No additional equipment needed (heat blocks, thermal cycler)

    • Cost per test of SDI less than BAX-PCR

    • SDI approved for use in place of PCR

    • Appropriate for use by labs analyzing a smaller number of samples


Value of Method Validation

  • Need to validate method on your intended product; rule out matrix interference

  • Determine minimum regulatory requirements (AOAC, AFNOR, etc)

  • Determine what is the right method for your lab based on volume of testing and number of technicians

  • Base selection of methodology on need

    • Sensitivity

    • Specificity

    • Speed

    • Cost

    • Lab space


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